Nutritional modulation of the host immune response to infection has been investigated in the critically ill with varied results, predominantly due to the heterogeneity of disease and differences in the composition of the dietary treatment. Understanding the function of individual compounds with respect to immunity would therefore be essential in evaluating the efficacy of dietary formulations. Dietary niacin undergoes several redox reactions resulting in the production of nicotinamide (NAM). Enzymes in the cytosol, mitochondria, and nucleus catalyze NAM transformation to NAD where the molecule is used in NAD(P)H redox reactions or consumed as a substrate by poly(ADP-ribose) polymerases (PARPs), CD38/CD157 ectoenzymes and sirtuins. Research has indicated that the disparate functions of myeloid cells may be linked to altered cell metabolism such that a pro-inflammatory (M1) macrophage utilizes anaerobic metabolism whereas an alternatively activated (M2) macrophage, involved in wound repair and tissue homeostasis, utilizes oxidative phosphorylation (OXPHOS) metabolism. The transcription factor HIF-1alpha is pivotal to anaerobic metabolism and a functional component in macrophage migration, phagocytosis, and antigen presentation. Macrophages isolated from HIF-1alpha deficient mice exhibit impaired clearance of gram positive and negative bacteria whereas pharmacological augmentation of HIF-1alpha boosts the response. Additional in vitro analysis of human monocyte derived macrophages (HMDMs) revealed that NAM antagonizes LPS-induced oxidative stress. These antioxidant properties of NAM decreased LPS-induced M1 pro-inflammatory cytokine production (tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-1beta) and increased components of an M2 phenotype (CD200 receptor and C-type mannose receptor 1 expression). In the field of immuno-oncology, blockade of the programmed death 1 (PD-1) axis is being examined as a means to induce anti-tumor immunity. Antibodies to PD-1 or PD-L1 are expected to block T cell PD-1 binding with its ligand (PD-L1, PD-L2) on antigen presentation cells or tumor cells and thereby antagonize T cell PD-1 cell signals thus encouraging tolerance. The cell surface receptor, CD38, binds NAD as well as the endothelial receptor CD31 and it additionally serves as a target for mainly B cell malignancies. In response to lipopolysaccharide (LPS), macrophages increase their expression of PD-L1 and CD38 on the cell surface, suggesting that NAM may also modify these responses. To begin to assess the functions of LPS and NAM with respect to metabolism, we examined if LPS could induce HIF-1alpha protein compared to a characterized hypoxia mimetic, DFOM, by immunoblot. Circulating monocytes from healthy volunteers were differentiated into HMDMs with 30 ng/ml M-CSF for 7 days. HMDMs (4x105 cells/ml) were incubated with 100 micro M DFOM or 10 ng/ml LPS for 24 hours. Cells were solubilized and immunoblots were probed for HIF-1alpha or actin. We identified a 120 kDa band associated with HIF-1alpha production that is up-regulated by DFOM and LPS compared to the control. To assess if NAM is able modulate the production of HIF-1alpha, we treated HMDMs with 10 ng/ml LPS in the absence or presence of increasing concentrations of NAM for 24 hours. We showed decreased production of HIF-1alpha in response to increasing concentrations of NAM compared to the LPS control. Intracellular signals in RAW cells stably transfected with reporters for p65 and TNF-alpha were additionally assessed for transcriptional activation after 24h incubation. NAM reduced LPS-induced reporter activity for both p65 and TNF-alpha. To further assess the functions of NAM, we examined characterized downstream effects of HIF-1alppha in HMDMs. We identified increased expression of CD80, CD40, and MHCI in the presence of LPS that is significantly reduced by NAM. These data suggest that NAM may alter innate and subsequent adaptive immune responses involving LPS. PD-1 interactions with its ligands, PD-L1 and PD-L2, modulate T cell/antigen presenting cell communications with negative regulatory effects. However, both PD-1 and PD-L1 have also been identified on subsets of macrophages in mediating responses to infection. PD-L1 is up-regulated by LPS and the expression is reduced significantly by NAM in HMDMs. We also identified PD-1 expression compared to an isotype control (data not shown) in HMDMs and noted that the receptor is not significantly modulated by LPS or NAM. Research has indicated that hypoxia enhances phagocytosis in macrophages in a HIF-1alpha-dependent manner. To initially assess HMDM phagocytosis, we examined the uptake of FITC-dextran. We showed that NAM significantly reduced HMDM uptake of dextran. To further understand phagocytic modifications by NAM in HMDMs, we examined the expression of mannose receptors (CD206, CD209) implicated in Pseudomonas aeruginosa phagocytosis. However, the identified expression levels of CD206 and CD209 across treatments and donors were variable and not significantly different (data not shown). We then tested the expression of CD38 which is a characterized macrophage activation marker and integral factor in FcgammaR-mediated phagocytosis. In fact, NAM significantly reduced LPS-induced expression of CD38. In summary, our preliminary studies have shown that in vitro treatment of human macrophages with NAM is able to reduce the expression of LPS-induced HIF-1alpha, MHCI, CD40, C80, PD-L1 and CD38. We have additionally found that macrophage uptake of FITC-labeled dextran is decreased by pre-treatment with NAM and the reporter activity of p65 and TNF-alpha, is also reduced in RAW cells. Our subsequent studies will focus on assessing metabolic shifts (ROS, lactate) and confirming the activity of p65 and HIF-1alpha as well as the modulation of cell surface molecules in human and murine macrophages in response to LPS and NAM after in vitro and in vivo treatment. In vivo challenge with a lethal dose of LPS in mice fed NAM supplemented or normal drinking water will also be assessed. These data are anticipated to reveal differences in the progression of pulmonary inflammation, particularly due to the distinct functions of hypoxia and PD-1/PD-L1 and CD38 in immunity and lung disease.